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Structure of a peptide-based photo-affinity cross-linker with Herceptin Fc
Structural highlights
FunctionPublication Abstract from PubMedSite-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein, we report the development and detailed characterization of a robust photoaffinity crosslinking method for site-specific conjugation to fully-glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously-described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. First, we describe the initial discovery of an effective Bpa mutant peptide and optimization of reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody. Second, we assessed the scope of the photoconjugation reaction across different human and non-human antibodies and antibody mutants. Third, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and show that the resulting conjugate is both stable in plasma and as potent as a conventional antibody drug conjugate in cells, portending well for future biological applications. Development, optimization and structural characterization of an efficient peptide-based photoaffinity crosslinking reaction for generation of homogeneous conjugates from wild-type antibodies.,Sadowsky J, Vance N, Zacharias N, Ultsch M, Li G, Fourie A, Liu P, Lafrance-Vanasse J, Ernst JA, Sandoval W, Kozak KR, Lewis-Phillips GD, Wang W Bioconjug Chem. 2018 Dec 19. doi: 10.1021/acs.bioconjchem.8b00809. PMID:30566343[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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